Neuronal damage in HIV-infected brain resulting in HIV-associated dementia (HAD) is thought to occur in part through oxidative stress-associated N-methyl-D-aspartate receptor (NMDAR) excitation initiated by HIV infection of brain macrophages. Brain regions enriched in NMDAR expressing the NR2A and NR2B subunits, such as the hippocampus, are particularly vulnerable to HIV. HIV proteins as well as glutamate, quinolinic acid (QUIN), platelet activating factor (PAF) and reactive oxygen species (ROS) released from activated glia can act as direct or indirect triggers for HIV-induced NMDAR-mediated excitotoxicity. In our initial funding period, we used our NT2.N neurotoxicity model to show that HIV-1-infected monocyte-derived macrophages (HIV/MDM) trigger the NMDAR-activated intrinsic (mitochondrial) apoptosis pathway. Furthermore, our recent study using our primary rodent hippocampal culture model demonstrates that HIV/MDM release excitotoxins that kill neurons by NMDAR-mediated calpain activation, and that hippocampal vulnerability is determined by NR2A and NR2B subunit expression. Our hypothesis is that HIV/MDM-induced NMDAR activation, altered NR subunit phosphorylation &localization are associated with macrophage stress responses and determine hippocampal neuronal vulnerability to HIV induced damage. Our studies of HIV/MDM neurotoxicity and the roles of NMDAR and the macrophage integrated stress response (ISR) have shown that i) hippocampal neuronal vulnerability is age-dependent and is determined by NR2A and NR2B expression patterns;ii) HIV/MDM rapidly induce neuronal calpain and cdk5 activation, altered NR2B phosphorylatioh &cleavage, and calpain- and cdk5-dependent death;iii) HIV replication in MDM induces the macrophage integrated stress response (ISR);and iv) the ISR is activated in macaque brain and human brain in individuals infected with immunodeficiency virus. To better define how HIV infection of macrophages triggers neurodegeneration cascades and to identify new targets for neuroprotection we will: 1) Define the pathways of calpain-induced NMDAR-mediated neurotoxicity induced by HIV/MDM and the functional roles for NR2 subunits;2) Determine whether specific phosphorylation and synaptic localization of NMDAR modulate HIV/MDM neurotoxicity and whether similar NR phosphorylation patterns are expressed in HAD;and 3) Determine the role of the macrophage Integrated Stress Response (ISR) in HIV/MDM-induced neurotoxicity. These studies offer appealing targets at multiple steps in the neurodegeneration cascade initiated by HIV infection that can potentially be exploited for identfication of currently available drugs and for the development of new neuroprotective strategies that can be effective against HIV-induced neurodegeneration. Project Narrative: This project investigates the mechanisms by which HIV-1 infection of macrophages induces neurodegeneration, using an in vitro infection model and pathological analyses of HIV-infected human brain and simian immunodeficiency virus (SIV)-infected macaque/monkey brain tissues. Our hypothesis, based on our preliminary studies, is that such infection alters the macrophage integrated stress response (ISR), leading to excitotoxin production and neuronal NMDA receptor activation by mechanisms that determine why selective neuronal populations are damaged in HIV-1 infection. We are attempting to validate our in vitro observations in human brain tissue and in the SIV macaque model so that neuroprotection studies can eventually be rationally designed for testing in the macaque model.